Annexins are a series of calcium ion-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiological conditions, PS is mainly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution on the phospholipid bilayer and can be transported to the outer membrane leaflet to mark the cell as a target for phagocytosis. While on the outer surface of the cell membrane, PS can be detected in a calcium-dependent mode by fluorescently labeled Annexin V.

In the early stages of apoptosis, the plasma membrane can block vitality dyes such as propidium iodide (PI) and 7-AAD or fixable vitality dyes such as eFluor 450 from entering the cell. These cells stain with annexin V but not with viability dyes, allowing differentiation of cells in early stages of apoptosis. However, in the late stages of apoptosis, the cell membrane loses its integrity, so annexin V can also access intracellular PS. Viability dyes can be used to distinguish these late apoptotic and necrotic cells (annexin V, viability dye positive) from early apoptotic cells (annexin V positive, viability dye negative).

Viability dyes currently provided with the eBioscience Annexin V Apoptosis Detection Kit include FITC, eFluor 450, allophycocyanin (APC), phycoerythrin (PE), PE-cyanine7 and biotin .